Download Cells To Tolerate Higher Expression of Bim T+ Bcl

Bcl-2 Allows Effector and Memory CD8+ T
Cells To Tolerate Higher Expression of Bim
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of August 2, 2017.
Sema Kurtulus, Pulak Tripathi, Maria E. Moreno-Fernandez,
Allyson Sholl, Jonathan D. Katz, H. Leighton Grimes and
David A. Hildeman
J Immunol 2011; 186:5729-5737; Prepublished online 30
March 2011;
doi: 10.4049/jimmunol.1100102
http://www.jimmunol.org/content/186/10/5729
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Supplementary
Material
The Journal of Immunology
Bcl-2 Allows Effector and Memory CD8+ T Cells To Tolerate
Higher Expression of Bim
Sema Kurtulus,* Pulak Tripathi,* Maria E. Moreno-Fernandez,† Allyson Sholl,*
Jonathan D. Katz,‡ H. Leighton Grimes,* and David A. Hildeman*
uring acute viral infection, naive CD8+ T cells expand
vigorously and generate a population of effector T cells.
Shortly after viral clearance, most of the effector T cells
die, whereas some remain and become memory T cells. Although
initial work suggested that Fas/Fas ligand signaling was critical
for the death of activated T cells (1–5), more recent work suggests
a dominant role for members of the Bcl-2 family (6). More specifically, the proapoptotic molecule Bim and its ability to signal
through the downstream proapoptotic executioners Bax and Bak
are essential for driving the contraction of T cell responses in vivo
(7–10). Failure to eliminate effector T cells in Bim-deficient animals leads to significantly enhanced T cell memory (10) and protective immunity (11). However, mechanism(s) by which some
cells succumb to, whereas others resist, Bim-driven death remain
unclear.
The effector CD8+ T cell response to acute viral infection is
heterogeneous, consisting of two major subsets, identified largely
D
*Division of Immunobiology, Department of Pediatrics, University of Cincinnati
College of Medicine and Children’s Hospital Medical Center, Cincinnati, OH 45229;
†
Division of Molecular Immunology, Department of Pediatrics, University of Cincinnati College of Medicine and Children’s Hospital Medical Center, Cincinnati, OH
45229; and ‡Division of Endocrinology, Department of Pediatrics, University of
Cincinnati College of Medicine and Children’s Hospital Medical Center, Cincinnati,
OH 45229
Received for publication January 13, 2011. Accepted for publication March 9, 2011.
This work was supported by Public Health Service Grant AI057753 (to D.A.H.).
Address correspondence and reprint requests to Dr. David A. Hildeman, Division of
Immunobiology, Department of Pediatrics, S5 Room 214, MLC 7038, Children’s
Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: CHX, cycloheximide; Foxo3a, forkhead box subclass O transcription factor 3a; gp33-sp, gp33-specific; KLRG1, killer cell lectin-like
receptor G1; LCMV, lymphocytic choriomeningitis virus; LCMV-sp, lymphocytic
choriomeningitis virus-specific; MFI, mean fluorescence intensity; p.i., postinfection;
TCM, central memory T cell; TEM, effector memory CD8+ T cell.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100102
by the reciprocal cell-surface expression of killer cell lectin-like
receptor G1 (KLRG1) and IL-7Ra (CD127) (1, 12, 13). Effector
CD8+ T cells with increased expression of CD127, but decreased
expression of KLRG1, survived more during contraction of the
response, and have been referred to as memory precursor effector
cells (12). Conversely, effector CD8+ T cells with decreased expression of CD127, but increased expression of KLRG1, survive less during contraction of the response and have been called
short-lived effector cells (12). The longer lifespan of KLRG1low
CD127high cells has been attributed to their increased expression
of the antiapoptotic molecule Bcl-2, although this has yet to be
tested experimentally.
Similar to effector T cells, heterogeneity also exists in the
memory compartment, as two major subpopulations of memory
T cells have been described (14). CD8+ effector memory T cells
(TEM) lack CD62L and CCR7 expression, and they reside in peripheral tissues (e.g., liver, lung, etc.) and the spleen. CD8+ central
memory T cells (TCM) expressing lymph node-homing receptors
CD62L and CCR7 are mostly found in the secondary lymphoid
organs. Although IL-7 and IL-15 critically control survival and
homeostatic turnover of overall memory T cells (15, 16), the role
of Bcl-2 in the survival of memory T cells is somewhat controversial. For example, Y449 in the cytoplasmic domain of IL-7Ra
was shown to be critical for survival of memory T cells but not for
IL-7–driven Bcl-2 upregulation (17), suggesting that antiapoptotic
signals other than Bcl-2 control memory T cell survival. In contrast, using a gene-knockout approach, we showed that Bcl-2 is
critical for IL-7– and IL-15–driven survival in vitro and for overall
memory T cell survival in vivo (18, 19). However, the requirement
of Bcl-2 in the survival of different subsets of memory CD8+
T cells was not determined in either of the previous studies (17,
18). Thus, the role of Bcl-2 in maintaining subsets of memory
T cells and the degree to which Bcl-2 combats Bim in such cells
remain unclear.
In naive T cells, most Bim is complexed to Bcl-2 (20), and we
recently showed that this interaction is critical to promote naive
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As acute infections resolve, most effector CD8+ T cells die, whereas some persist and become memory T cells. Recent work showed
that subsets of effector CD8+ T cells, identified by reciprocal expression of killer cell lectin-like receptor G1 (KLRG1) and CD127,
have different lifespans. Similar to previous reports, we found that effector CD8+ T cells reported to have a longer lifespan
(i.e., KLRG1lowCD127high) have increased levels of Bcl-2 compared with their shorter-lived KLRG1highCD127low counterparts.
Surprisingly, we found that these effector KLRG1lowCD127high CD8+ T cells also had increased levels of Bim compared with
KLRG1highCD127low cells. Similar effects were observed in memory cells, in which CD8+ central memory T cells expressed higher
levels of Bim and Bcl-2 than did CD8+ effector memory T cells. Using both pharmacologic and genetic approaches, we found that
survival of both subsets of effector and memory CD8+ T cells required Bcl-2 to combat the proapoptotic activity of Bim.
Interestingly, inhibition or absence of Bcl-2 led to significantly decreased expression of Bim in surviving effector and memory
T cells. In addition, manipulation of Bcl-2 levels by IL-7 or IL-15 also affected expression of Bim in effector CD8+ T cells. Finally,
we found that Bim levels were significantly increased in effector CD8+ T cells lacking Bax and Bak. Together, these data indicate
that cells having the highest levels of Bim are selected against during contraction of the response and that Bcl-2 determines the
level of Bim that effector and memory T cells can tolerate. The Journal of Immunology, 2011, 186: 5729–5737.
5730
T cell survival (18). Following activation, levels of Bcl-2 within
activated T cells are decreased, which is correlated with enhanced
death of effector T cells (21). Recent work, however, has suggested a potentially more complex interplay between Bim and
Bcl-2 (22), as it was shown that genetic ablation of Bim leads to
decreased levels of Bcl-2 protein, whereas overexpression of Bcl-2
promotes increased expression of Bim mRNA and protein within
activated T cells (21, 22). These data suggested that Bim and Bcl2 could reciprocally affect the other’s expression in T cells, although the mechanism(s) underlying such phenomenon remain
unclear.
In this study, we examined the role of Bim and Bcl-2 in both
effector and memory subsets of CD8+ T cells using both pharmacologic and genetic approaches. We found that both effector
and viral-specific memory CD8+ T cells require Bcl-2 to combat
the proapoptotic effects of Bim and, by doing so, determine the
levels of Bim CD8+ T cells can tolerate and survive.
Mice and viral infection
C57BL/6 mice were either purchased from The Jackson Laboratory or
Taconic Farms. Bim2/2 mice were a gift from P. Bouillet and A. Strasser
(Walter and Eliza Hall Institute, Melbourne, VIC, Australia) and have been
backcrossed to C57/BL6 mice for at least 14 generations. Breeding pairs of
Bcl-2Tm1sjk mice were obtained from The Jackson Laboratory and were
mated to Bim2/2 mice to generate Bim+/2Bcl-22/2, Bim+/2Bcl-2+/2, and
Bim2/2Bcl-22/2 mice. Lck Cre2Baxf/fBak2/2 mice were a gift from Dr.
Stanley Korsmeyer. IL-15–deficient mice on a C57BL/6 background were
purchased from Taconic Farms. ABT-737 was a generous gift of Abbott
Laboratories, dissolved in DMSO, and diluted in 35% polyethylene glycol,
5% Tween-80, and 65% of dextrose in water solution. Mice were injected
i.p. once a day with 75 mg/kg in a volume of 0.2 ml. Mice were infected
i.p. with 2 3 105 PFU lymphocytic choriomeningitis virus (LCMV).
LCMV was grown in BHK-21 cells, and viral titers from spleen and liver
homogenates were determined by plaque assay on BHK-21 monolayers as
described. For BrdU incorporation, mice were injected i.p. with three doses
of 0.7 mg/mouse BrdU (BD Biosciences, San Jose, CA) 2 d before sacrifice (one on day 22 and two on day 21). BrdU incorporation was assessed with the BrdU Flow kit (BD Biosciences, San Jose, CA) according
to the manufacturer’s instructions. Animals were housed under specific
pathogen-free conditions in the Division of Veterinary Services at Cincinnati Children’s Hospital Research Foundation. Experimental procedures
were reviewed and approved by the Institutional Animal Care and Use
Committee at the Cincinnati Children’s Hospital Research Foundation.
Retroviral transduction
C57BL/6 mice were injected with 100 mg Staphylococcus enterotoxin B
(Toxin Technology, Sarasota, FL), and 1 d later, T cells were isolated by
MACS isolation (Miltenyi Biotec, Auburn, CA). Activated T cells were
spin-transduced with empty vector (MIT) or MIT–Bcl-2 retroviruses at
room temperature for 2 h. Retroviruses were produced as previously described (21). Transduced T cells were stained with Abs against Thy1.1,
Vb8, CD4, CD8, and Bim and analyzed by flow cytometry.
In vitro treatments
Splenocytes from either uninfected or day 10 LCMV-infected mice were
cultured with 2 mM cycloheximide (CHX) (Sigma-Aldrich, St. Louis, MO)
for 6 h at 37˚C. To reduce variability in staining, cells were harvested after
culture and stained alongside cells that had not been cultured to assess the
decline of Bim protein from time zero.
Quantitative real-time PCR
Spleen cells from LCMV-infected mice on day 20 p.i. were harvested and
stained with CD8, CD44, CD62L, KLRG1, and CD127 Abs. CD44+
CD62L2KLRG1highCD127low or CD44 +CD62L2KLRG1low CD127high
CD8+ T cells from C57BL/6 or Lck-Cre+Baxf/fBak2/2 mice were sorted
with FACSAria (BD Biosciences), and RNA was isolated from cells using
Qiagen’s RNeasy Mini Isolation kit (Qiagen) and converted into cDNA
using Superscript II Reverse transcriptase (Invitrogen, CA). Real-time
PCR was performed with IQ SyBrGreen Supermix (Bio-Rad, Hercules,
CA), and primers for Bim were 59-ACAAACCCCAAGTCCTCCTT-39 and
59-GTTTCGTTGAACTCGTCTCC-39; and for internal control L19: 59CCTGAAGGTCAAAGGGAATGTG-39 and 59-GCTTTCGTGCTTCCTTGGTCT-39. Real-time PCR was performed in IQ Cycler PCR Machine
(Bio-Rad).
Results
Levels of Bcl-2 and Bim are correlated in subpopulations of
effector CD8+ T cells
Spleens from individual mice were harvested and crushed through a 70-mm
mesh cup (BD Falcon) to generate single-cell suspensions. A total of 2 3
106 cells were stained with different combinations of the following cellsurface Abs: anti-CD8, CD44, CD62L, KLRG1, CD127 (from either Biolegend or eBioscience, San Diego, CA) and intracellularly with either anti–
Bcl-2 (clone 3F11, produced in-house) or anti-Bim (Cell Signaling Technology, Beverly, MA) as described. Dbgp33 monomers were produced in
collaboration with Dr. Aaron Johnson and were coupled to either APC or
PE as previously described (10). A minimum of 5 3 105 events was acquired on a BD LSR II flow cytometer and analyzed by either FACSDiva
(BD Biosciences) or FlowJo software (Tree Star).
Although the increased survival of KLRG1lowCD127high effector
CD8+ T cells was attributed to their increased expression of Bcl-2,
this was not tested (12). In addition, as Bcl-2 is critical to combat
Bim to ensure naive T cell survival (18), and Bim is critical for
contraction of T cell responses (10, 21), it is possible that decreased levels of Bim could contribute to enhanced survival of
KLRG1lowCD127high effector CD8+ T cells. Using a mouse model
of LCMV infection, we examined the levels of Bim and Bcl-2
within LCMV-specific (LCMV-sp) effector CD8+ T cell subsets
by intracellular flow cytometry using specific Abs (23). First, we
found that the levels of Bim and Bcl-2 were largely correlated in
both KLRG1highCD127low and KLRG1lowCD127high subsets of
effector Dbgp33-specific (gp33-sp) CD8+ T cells at the peak of the
response (Fig. 1A). Next, we found that Bcl-2 levels were significantly higher in KLRG1lowCD127high cells compared with
KLRG1highCD127low effector Dbgp33-sp CD8+ T cells 10 d p.i.
(Fig. 1B, 1C). Surprisingly, we also found that Bim levels were
significantly increased in KLRG1 lowCD127high effector CD8+
T cells (Fig. 1B, 1C). Thus, both Bim and Bcl-2 are more highly
expressed in effector CD8+ T cells with more potential to develop
into long-lived memory cells.
IL-7 and IL-15 manipulation in vivo
Bcl-2 is critical to combat Bim for survival of KLRG1low
CD127high effector CD8+ T cells
For in vivo IL-7 blockade experiments, M25 was grown as ascites, purified
by ammonium sulfate precipitation and ion exchange chromatography, and
injected i.p. at a dose of 3 mg/mouse every other day. Effectiveness of IL-7
blockade was assessed by measuring the numbers of pre-B cells in the bone
marrow of each mouse via flow cytometry using Abs against IgM, B220, and
CD24. For IL-7 and IL-15 delivery experiments, human rIL-7/anti–IL-7
immune complexes were mixed in vitro, and the equivalent of 2.5 mg
human rIL-7 (PeproTech, Rocky Hill, NJ) was injected i.p. on days 8, 10,
12, and 14 postinfection (p.i.). For IL-15 delivery experiments, IL-15/IL15Ra (R&D Systems, Minneapolis, MN) was mixed in vitro, and the
equivalent of 750 ng IL-15 was injected i.p. on days 8, 10, 12, and 14 p.i.
We next used both genetic and pharmacological approaches to
determine whether Bcl-2 is critical for the survival of effector CD8+
T cells following LCMV infection. First, we took advantage of
Bim+/2Bcl-22/2 mice, as loss of a single allele of Bim has previously been shown to prevent the early lethality and kidney
disease of Bcl-2 deficiency (24). We infected groups of C57BL/6
and Bim+/2Bcl-22/2 mice with LCMV and analyzed the effector
CD8+ T cell subsets 10 and 23 d later. As expected, ∼75–80%
of KLRG1highCD127low and ∼30–40% of KLRG1lowCD127high
MHC tetramer staining and flow cytometry
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Materials and Methods
Bim/Bcl-2 BALANCE AND EFFECTOR CD8+ T CELLS
The Journal of Immunology
effector gp33-sp CD8+ T cells were lost between days 10 and 23
p.i. (Fig. 2A). Interestingly, deficiency in Bcl-2 did not exacerbate
the loss of KLRG1highCD127low cells, but it significantly enhanced the loss of KLRG1lowCD127high cells (Fig. 2A). This
greater loss of effector CD8+ T cells in the absence of Bcl-2 was
largely alleviated by the additional loss of the remaining allele of
Bim (Fig. 2A). These data show that Bcl-2 is critical to counteract
Bim in KLRG1lowCD127high effector CD8+ T cells.
As the lymphopenic environment and potential developmental
abnormalities in T cells from Bim+/2Bcl-22/2 mice could have
impacted the above results, we took advantage of a synthetic Bcl-2
antagonist, ABT-737, that inhibits Bcl-2, Bcl-xL, and Bcl-w (25).
Our previous data showed that ABT-737 kills naive T cells in
C57BL/6 mice in a Bim-dependent manner (18). To determine the
role of Bcl-2 in effector T cell survival, we treated C57BL/6 mice
with ABT-737 or vehicle between days 10 and 23 after LCMV
FIGURE 2. Bcl-2 is critical to combat Bim for survival of KLRG1low
CD127high effector CD8+ T cells. A, Groups of C57BL/6, Bim+/2Bcl-22/2,
and Bim2/2Bcl-22/2 mice (n = 4 mice/group) were infected i.p. with 2 3
105 PFU/mouse LCMV and sacrificed 10 or 23 d p.i. Splenocytes were
stained with Dbgp33 tetramers and Abs against KLRG1, CD127, and CD8.
CD8+GP33+ KLRG1lowCD127high or CD8+GP33+ KLRG1highCD127low
populations were analyzed on days 10 or 23 after LCMV infection by flow
cytometry. Bar graphs show the percentage decrease in the numbers of
each CD8+ T cell effector subset 6 SEM from day 10 to day 23 p.i. B,
C57BL/6 mice were infected i.p. with LCMV. Mice were treated either
with vehicle or the synthetic Bcl-2 inhibitor ABT-737 (1 mg/mouse/d) (n =
4 mice/group) between days 10 and 23 p.i. Mice were sacrificed 23 d p.i.,
and splenocytes were stained as in A. Bar graphs show the percentage
decrease in the numbers of each CD8+ T cell effector subset 6 SEM from
days 10–23 p.i. Data are representative of two independent experiments. C,
C57BL/6 mice were infected i.p. with LCMV. Mice (n = 9 mice/group)
were treated either with vehicle or the synthetic Bcl-2 inhibitor ABT-737
between days 10 and 20 p.i as in B. Mice were sacrificed 110 d p.i., and
splenocytes were stained with Dbgp33 tetramers and Abs against CD8,
CD62L, and CD44. CD8+ TEM and TCM numbers 6 SEM values are
shown. Data are pooled from two independent experiments. *p # 0.05.
infection. Pharmacological inhibition of Bcl-2 resulted in a significant reduction in the numbers of gp33-sp KLRG1lowCD127high
cells, as 90% of these cells were lost during the contraction phase
compared with an ∼45% reduction in vehicle-treated mice (Fig.
2B). In contrast, loss of KLRG1highCD127low cells was slightly
exacerbated by Bcl-2 inhibition (Fig. 2B). Thus, similar to our
genetic studies, pharmacologic inhibition of Bcl-2 also resulted in
a significant loss of effector CD8+ T cells, particularly affecting
KLRG1lowCD127high cells.
Bcl-2 is critical for the generation and survival of memory
CD8+ T cells
As KLRG1lowCD127high CD8+ effector T cells contribute to the
long-term memory cell pool (12, 13), we next determined how
inhibition of Bcl-2 during the contraction phase would affect
memory development. Again, we treated C57BL/6 mice with
ABT-737 between day 10 and 20 after LCMV infection and analyzed gp33-sp CD8+ memory T cells 110 d later. We found that
the overall numbers of TCM and TEM were decreased in ABT-737–
treated mice compared with those treated with vehicle (Fig. 2C).
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FIGURE 1. Levels of Bcl-2 and Bim are correlated in subpopulations of
effector CD8+ T cells. C57BL/6 mice (n = 4 mice) were infected with 2 3
105 PFU/mouse LCMV Arm strain i.p. and sacrificed on either day 8 (A) or
10 (B) p.i. Cells from Bim2/2 and Bim+/2Bcl-22/2 mice were included as
Ab specificity controls in experiments in B. Single-cell spleen suspensions
were stained with Dbgp33 tetramers and Abs against KLRG1, CD127, and
CD8 and intracellularly against Bim and Bcl-2 and data acquired on a flow
cytometer. A, Representative dot plots show Bim and Bcl-2 levels in
subpopulations of CD8+ Dbgp33+ cells gated as KLRG1lowCD127high or
KLRG1highCD127low cells. B, Representative histograms of Bim and Bcl-2
in KLRG1lowCD127high or KLRG1highCD127low cells from either C57BL/
6 mice (filled histograms) or Bim2/2 (open Bim histograms), Bim+/2Bcl22/2 mice (open Bcl-2 histograms) are shown. C, Bar graphs show the
mean fluorescence intensity (MFI) values 6 SEM for Bim and Bcl-2 in
KLRG1highCD127low or KLRG1lowCD127high cells from C57BL/6 mice on
day 10 p.i. Data are pooled from two independent experiments including
the one shown in B. p values for statistically significant differences were
calculated by Student t test. **p # 0.01.
5731
5732
A
B
C
D
FIGURE 4. Bcl-2 is critical for survival of memory CD8+ T cells.
Groups of C57BL/6 and Bim2/2 mice (n = 5 mice/group) were infected
i.p. with LCMV, and after 90 d, they were treated with either vehicle or
ABT-737 (1 mg/mouse/d) for 10 d and sacrificed. Mice were also injected
with BrdU (0.7 mg/mouse i.p.) for 2 d before sacrifice. Spleen cells
were stained with Dbgp33 tetramers and Abs against CD8, CD44, and
CD62L and analyzed by flow cytometry. A, Bar graphs indicate the total
numbers 6 SEM values of TEM (CD8+CD44highCD62Llow) or TCM (CD8+
CD44highCD62Lhigh) subsets. B, Splenocytes were stained with the above
MHC tetramer and surface stains along with an Ab against BrdU according
to the manufacturer’s instructions. The percent of TEM and TCM that were
BrdU+ 6 SEM are shown. C, Splenocytes were also stained with the above
MHC tetramer and surface stains along with an Ab against Bim. Bar
graphs show Bim MFI 6 SEM values in TEM and TCM subsets. Graphs are
representative of three independent experiments. D, Groups of C57BL/6,
Bim+/2Bcl-22/2, and Bim+/2Bcl-2+/2 mice (n = 4 mice/group) were
infected i.p. with LCMV and sacrificed 100 d p.i. Splenocytes were stained
with MHC tetramers and surface stains as above and intracellularly for
Bim. Bar graphs show Bim MFI 6 SEM values in TEM and TCM subsets.
*p # 0.05, **p # 0.01.
FIGURE 3. Expression of Bim and Bcl-2 in memory CD8+ T cell
subpopulations. C57BL/6 mice were infected i.p. with LCMV. Mice (n = 6
mice/group) were sacrificed 100 d later, and splenocytes were stained with
Dbgp33 tetramers and Abs against CD8, CD44, CD62L, and intracellularly
against Bim or Bcl-2, and data were acquired on a flow cytometer and
analyzed using FACSDiva software after gating on TEM (CD8+CD44high
CD62Llow) (TEM) or TCM (CD8+CD44highCD62Lhigh) populations. A, Dot
plots for Bim and Bcl-2 in TEM and TCM after gating on CD8+GP33+ cells.
Histograms (B) and bar graphs (C) for Bim and Bcl-2 MFI in TEM or TCM
CD8+ T cells are shown. Graphs are representative of three independent
experiments. **p # 0.01.
critical for the maintenance of LCMV-sp memory cells. As ABT737 can cause moderate lymphopenia (18) that can in turn cause
homeostatic proliferation, we assessed in vivo T cell proliferation
using BrdU. gp33-sp CD8+ TCM and, to a lesser extent, TEM in
ABT-737–treated C57BL/6 mice underwent significantly greater
proliferation in vivo (Fig. 4B). ABT-737 did not increase proliferation of gp33-sp CD8+ T cells in Bim2/2 mice (Fig. 4B), as
Bim2/2 mice are resistant to ABT-737–induced lymphopenia (18).
Thus, the actual dependence of viral-specific memory T cells upon
Bcl-2 is likely underestimated because of the lymphopenia-induced
proliferation following ABT-737 treatment.
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Thus, these results suggest that generation of normal numbers of
memory CD8+ T cells critically depends upon the function of Bcl2 during the contraction of the response.
Although inhibiting Bcl-2 at the effector stage can have a longterm impact on CD8+ T cell memory, we next determined the roles
and expression levels of Bim and Bcl-2 in memory T cells after
they were formed. First, we measured Bim and Bcl-2 levels in
gp33-sp TEM and TCM CD8+ T cells by flow cytometry. Similar to
subsets of effector CD8+ T cells, the levels of Bim and Bcl-2 were
directly correlated in both TEM and TCM subsets of CD8+ gp33-sp
memory cells (Fig. 3A). Further, levels of both Bim and Bcl-2
were significantly increased in TCM compared with TEM within
the CD8 + gp33-sp population (Fig. 3B, 3C). Thus, similar to
KLRG1lowCD127high cells, gp33-sp TCM CD8+ T cells exhibited
high expression of both Bim and Bcl-2.
Next, we determined the role of Bcl-2 in combating Bim to
maintain LCMV-sp memory CD8+ T cells. We infected C57BL/6
and Bim2/2 mice with LCMV and, 90 d later, treated them with
ABT-737 or vehicle control for 10 d and analyzed gp33-sp memory cell subsets by flow cytometry. In C57BL/6 mice, ABT-737 led
to a ∼3-fold loss of gp33-sp CD8+ TCM and a ∼2-fold loss of TEM
cells (Fig. 4A). As expected, numbers of TEM and TCM in vehicletreated Bim-deficient mice were significantly increased compared
with vehicle-treated C57BL/6 mice (Fig. 4A). Further, gp33-sp TEM
and TCM in Bim-deficient mice were not significantly decreased
by ABT-737 (Fig. 4A), suggesting that a Bim/Bcl-2 balance is also
Bim/Bcl-2 BALANCE AND EFFECTOR CD8+ T CELLS
The Journal of Immunology
5733
Bcl-2 determines the level of Bim in effector and memory
CD8+ T cells
IL-7 and IL-15 availability can influence the levels of Bim in
effector CD8+ T cells
It is well known that IL-7 and IL-15 (as well as other common
g-chain cytokines) can increase expression of Bcl-2 within effector T cells (15, 26–28). Therefore, we predicted that manipulation of IL-7 and/or IL-15 levels during contraction of the CD8+
T cell response would affect their levels of Bim in a manner that
correlates with Bcl-2 expression. First, we examined Bim levels in
effector T cells from LCMV-infected C57BL/6 and IL-15–deficient mice that were treated with either isotype control Ab or
anti–IL-7 neutralizing Ab between days 10 and 20 p.i. We found
FIGURE 5. Bcl-2, IL-7, and IL-15 determine the level of Bim in effector
CD8+ T cells. A and B, Groups of C57BL/6, Bim+/2Bcl-22/2, and Bim2/2
Bcl-22/2 mice (n = 4 mice/group) were infected i.p. with LCMV and
sacrificed 10 or 23 d p.i. Splenocytes were stained with Dbgp33 tetramers
and Abs against KLRG1, CD127, CD8, and intracellularly for Bim. Bar
graphs show Bim MFI 6 SEM values in KLRG1highCD127low (A) and
KLRG1lowCD127high (B) subsets. Significant differences were observed
for C57BL/6 mice between days 10 and 23 and between Bim+/2Bcl-2+/2
and Bim+/2Bcl-22/2 mice on day 23 (A) and for Bim+/2Bcl-22/2 mice
between days 10 and 23 (B). C, Groups of C57BL/6 or IL-152/2 mice (n =
5 mice/group) were infected with LCMV and treated i.p. with either isotype control (MPC11) or anti–IL-7 (M25) Ab (3 mg/mouse) on days 11,
13,15, 17, and 19 and sacrificed on day 20. Splenocytes were stained with
Dbgp33 tetramers and Abs against KLRG1, CD8, and Bim. Bar graphs
show Bim MFI 6 SEM values in KLRG1high and KLRG1low subsets. D,
C57BL/6 mice (n = 4 mice/group) were infected with LCMV, treated with
PBS, IL-7/anti–IL-7 complexes, or IL-15/IL-15Ra complexes on days 8,
10, 12, and 14 p.i., and sacrificed on day 16 p.i. Splenocytes were stained
with Dbgp33 tetramers and Abs against CD127, CD8, and Bim. Bar graphs
show Bim MFI 6 SEM values in CD127low and CD127high subsets. *p #
0.05, **p # 0.01.
that, in both KLRG1high and KLRG1low cells, neutralization of IL7 or loss of IL-15 led to decreased levels of Bim, which was
slightly, albeit nonsignificantly, decreased by neutralization of IL7 in IL-152/2 mice (Fig. 5C). Recently, we showed that IL-7 and
IL-15 are critical for maintaining expression of Bcl-2 in effector
CD8+ T cells (19). Thus, IL-7 and IL-15 are critical to maintain
expression of both Bcl-2 and Bim in effector CD8+ T cells.
Conversely, we reasoned that administration of IL-7 or IL-15,
either of which can increase Bcl-2 levels in effector T cells (19,
26, 29), would also increase their expression of Bim. To test this,
Downloaded from http://www.jimmunol.org/ by guest on August 2, 2017
As Bim and Bcl-2 levels within effector and memory CD8+ T cells
are correlated and have been shown to influence expression of
each other (21, 22), we next determined the role of Bcl-2 in maintaining Bim expression in memory T cells. To do this, we first
treated C57BL/6 mice infected with LCMV 3 mo previously with
ABT-737 or vehicle. Interestingly, we found that Bim levels were
significantly decreased in both memory CD8+ subsets from ABT737–treated compared with controls (Fig. 4C). We also assessed
the influence of Bcl-2 on Bim levels in gp33-sp CD8+ T cells in
groups of LCMV-infected C57BL/6, Bim+/2Bcl-2+/2, and Bim+/2
Bcl-22/2 mice. Bim levels in CD8+ T cells from both Bim+/2Bcl2+/2 and Bim+/2Bcl-22/2 mice should be ∼50% of that found in
C57BL/6 mice, unless the lack of Bcl-2 has an effect on Bim
levels. Similar to the results with ABT-737, we found that the
levels of Bim within both gp33-sp TEM and TCM subpopulations of
memory cells from Bim+/2Bcl-22/2 mice were significantly decreased compared with cells from either C57BL/6 or Bim+/2Bcl2+/2 mice (Fig. 4D). Thus, using both pharmacologic and genetic
approaches, we found that in TCM, and to a lesser extent TEM, Bcl2 is critical to maintain significant expression of Bim.
As Bim levels were decreased in TEM and TCM in Bim+/2Bcl2/2
2
mice and because these mice have a substantial loss of effector T cells, we next inquired whether the low levels of Bim in
their memory cells were apparent earlier, during either the peak or
after contraction of the response. To do this, we examined the
levels of Bim within gp33-sp effector CD8+ T cells on days 10 and
23 after LCMV infection. In C57BL/6 mice, expression of Bim
within gp33-sp CD8+ KLRG1highCD127low cells was significantly
decreased from days 10–23 p.i. (Fig. 5A). In contrast, Bim levels
remained high in the KLRG1lowCD127high subset on days 10 and
23 p.i. (Fig. 5B).
Next, we determined the role of Bcl-2 in influencing the levels
of Bim in gp33-sp effector CD8+ T cell subsets. Using LCMVinfected C57BL/6 mice, Bim+/2Bcl-2+/2, and Bim+/2Bcl-22/2
mice, we assessed Bim levels within effector CD8+ subsets. On
day 10 p.i., the levels of Bim in both KLRG1highCD127low and
KLRG1lowCD127high effector cells in both Bim+/2Bcl-2+/2 and
Bim+/2Bcl-22/2 mice were roughly half of that observed in
C57BL/6 mice (Fig. 5A, 5B). Interestingly, on day 23 p.i., the
levels of Bim decreased slightly in KLRG1highCD127low cells
from Bim+/2Bcl-2+/2 mice, but decreased to a significantly greater
extent in cells from Bim+/2Bcl-22/2 mice (Fig. 5A, 5B). Moreover,
in KLRG1lowCD127high cells, the levels of Bim were only significantly reduced in mice that completely lacked expression of Bcl-2
(Fig. 5A, 5B). Similar decreases in Bim in effector CD8+ T cells
were observed when C57BL/6 mice were treated with ABT-737
during contraction of the response (data not shown). Thus, Bcl-2
is required to maintain normal levels of Bim within effector
CD8+ T cells.
5734
we infected groups of C57BL/6 mice with LCMV and administered PBS, human IL-7/anti–IL-7, or IL-15/IL-15Ra complexes
on days 8, 10, 12, and 14 p.i., sacrificed them on day 16 p.i., and
measured Bim levels within effector CD8+ T cells. In contrast to
inhibition of IL-7 and IL-15, administration of either IL-7 or IL-15
significantly increased levels of Bim within effector CD8+ T cells
(Fig. 5D). Thus, enhancing IL-7 or IL-15 availability increases the
levels of both Bim and Bcl-2 in effector CD8+ T cells.
Posttranslational control of Bim by Bcl-2
induced loss of Bim protein was similar between KLRG1high
CD127low and KLRG1lowCD127high effector CD8+ T cells (Fig.
6A). We next used Bim+/2Bcl-22/2 mice to test the role of Bcl-2
in stabilization of Bim protein. We found that the lack of Bcl-2
slightly, albeit significantly, exacerbated the CHX-induced loss
of Bim in KLRG1 low CD127high effector cells compared with
C57BL/6 and Bim+/2 controls (Fig. 6B). In contrast, the absence
of Bcl-2 did not accelerate the loss of Bim in KLRG1highCD127low
effector cells. Thus, Bcl-2 likely contributes somewhat to Bim
stabilization, at least in KLRG1lowCD127high effector cells.
Lack of cell death allows effector T cells to tolerate higher
expression of Bim
Another potential, and not mutually exclusive, explanation is that
a threshold level of Bcl-2 is critical to protect effector CD8+ T cells
from toxic levels of Bim. A prediction of this model is that inhibition of apoptosis should lead to increased expression of Bim.
We tested this prediction using two separate approaches. First,
we determined whether overexpression of Bcl-2, which protects
activated T cells from death (21), would affect Bim levels in activated T cells. Similar to a recent report (22), retroviral overexpression of Bcl-2 led to significantly increased levels of Bim
FIGURE 6. Bcl-2 regulates the amount of Bim effector CD8+ T cells can tolerate. A, C57BL6 mice (n = 3 mice) were infected i.p. with LCMV. Mice
were sacrificed 10 d p.i. together with uninfected C57BL/6 mice. Splenocytes were cultured with or without 2 mM CHX for 6 h and then stained with Db
gp33 tetramers and Abs against CD8, KLRG1, CD127, and Bim. Bar graphs show the percentage change in Bim MFI levels 6 SEM values in CHX-treated
cells relative to uncultured cells. B, Groups of C57BL/6, Bim+/2, and Bim+/2Bcl-22/2 mice (n = 3 mice/group) were infected i.p. with LCMV and
sacrificed 10 d p.i. Splenocytes were cultured with or without 2 mM CHX for 6 h and then stained as in A. Bar graphs show the percentage change in Bim
MFI levels 6 SEM values in CHX-treated cells relative to uncultured cells. C–F, Groups of C57BL/6, Lck Cre2Baxf/f Bak2/2 and Lck Cre+Baxf/fBak2/2
mice (n = 3–6 mice/group) were infected i.p. with LCMV and sacrificed on days 20 and 23 p.i. Data in C, E, and F are pooled from two independent
experiments. C, E, and F, Splenocytes were stained with Dbgp33 tetramers and Abs against KLRG1, CD127, and CD8 and intracellularly for Bim (C) and
Bcl-2 (E). C, Bar graphs show Bim MFI 6 SEM values in KLRG1highCD127low and KLRG1lowCD127high subsets. D, Real-time PCR for Bim mRNA from
CD44+CD62L2KLRG1highCD127low or CD44+CD62L2KLRG1lowCD127high cells that were FACS sorted from pooled C57BL/6 mice or three Lck Cre+
Baxf/fBak2/2 individual mice on day 23 p.i. Expression levels were normalized to L19 expression. Similar results were obtained in sorted cells on day
20 p.i. E, Bar graphs show Bcl-2 MFI 6 SEM values in KLRG1highCD127low and KLRG1lowCD127high subsets. F, Bar graphs show the total numbers of
each CD8+ T cell effector subset 6 SEM. **p # 0.01.
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As Bcl-2 is complexed to Bim in naive T cells (20), it is possible
that Bcl-2 influences the levels of Bim through protein–protein
interactions that increase the stabilization of Bim (30, 31). We
next measured Bim turnover in effector CD8+ T cells by culturing
splenocytes from either uninfected or day 10 LCMV-infected
C57BL/6 mice with the inhibitor CHX and assessed Bim protein
levels over time in culture. We found that 6 h was a reasonable
time to assess Bim turnover because: 1) there was a significant
loss of Bim at this time point; and 2) there was little cell death
occurring during this time (data not shown). After culture with
CHX, levels of Bim decreased moderately in naive T cells and
substantially in effector T cells (Fig. 6A). Further, the CHX-
Bim/Bcl-2 BALANCE AND EFFECTOR CD8+ T CELLS
The Journal of Immunology
Discussion
We and others previously showed that most effector CD8+ T cells
have decreased expression of Bcl-2 at the peak of the response
(21, 26, 32). Further, our previous data in a superantigen model
showed that decreased expression of Bcl-2 correlated with susceptibility to Bim-driven death of effector T cells (21). More recently, it was found that subsets of effector T cells differ in their
expression of Bcl-2 (13, 33). In addition, it is well documented
that most memory T cells express high levels of Bcl-2 (32).
However, in none of these reports was the actual role of Bcl-2 in
the survival of effector and memory T cells tested. We recently
found that Bcl-2 was critical for the survival of most naive and
some memory T cells (18). In this study, we show that Bcl-2
is critical for the survival of particular subsets of effector and
memory T cells and, in doing so, allowed these cells to tolerate
higher levels of Bim and survive.
A recent report showed that Bim and Bcl-2 can reciprocally
affect the expression of each other (22), although the underlying
mechanism(s) were unclear. Similar to our results, Marrack’s
group (22) found that retroviral overexpression of Bcl-2 led to
increased levels of Bim mRNA and protein within activated
T cells. In this study, we found that Bcl-2 was important to prevent
the loss of Bim protein in effector T cells, likely acting at two
levels. At one level, Bcl-2 likely stabilizes Bim, a normally unstructured protein (34), and likely protects it from proteasomal
degradation. At another level, a consequence of Bcl-2 binding to
Bim is the prevention of cell death, which allows cells with higher
levels of Bim to survive. Combined, these data suggest a model in
which, following T cell activation, Bim levels are transcriptionally
increased, but most effector T cells do not maintain sufficient
levels of Bcl-2 to protect these T cells from death. The few cells
that do maintain Bcl-2 at high levels also appear to have the
highest levels of Bim; elimination or inhibition of Bcl-2 in these
cells leads to rapid death, and only cells having the lowest expression of Bim survive. Conversely, inhibition of apoptosis
downstream of Bim (i.e., in effector T cells that lack Bax and Bak)
allowed cells to survive despite having substantially higher Bim
mRNA and protein levels. This Bim/Bcl-2 balance in the effector stage is absolutely critical to memory T cell development, as
disruptions to this balance affect the generation of normal numbers of memory CD8+ T cells.
Although the loss of Bcl-2 in effector T cells is likely a significant initiating apoptotic event, our data also suggest an activationinduced increase in Bim expression; this has been difficult to
observe previously, because the cells die. Indeed, when this death
was eliminated by genetic deletion of Bax and Bak, we saw
a significant increase in Bim mRNA and protein in both subsets of
effector CD8+ T cells. Interestingly, even though death was alleviated, and Bim levels were increased in both effector CD8+ T cell
subsets, they were not increased to the same overall level
(i.e., Bim levels were still lower in KLRG1highCD127low cells). It
is unlikely that different levels of Bcl-2 in the different subsets
of CD8+ T cells in Bax/Bak double-deficient mice contribute to
this differential expression of Bim, because in both effector subsets, Bcl-2 levels are significantly lower than those observed
in C57BL/6 animals. In contrast, the transcriptional profiles of
KLRG1highCD127low and KLRG1lowCD127high cells have been
reported to be different (12). It is possible that Bim expression is
controlled by distinct mechanisms in KLRG1highCD127low and
KLRG1lowCD127high cells.
We and others have recently shown that KLRG1highCD127low
cells are predominantly maintained by IL-15, whereas IL-7 and
IL-15 act redundantly to maintain KLRG1lowCD127high cells (19,
35). We also found that Bcl-2 is a critical antiapoptotic molecule
induced/maintained by IL-7 and IL-15 in both of these subsets
(19). In this study, we show that Bim levels are also regulated by
the availability of IL-7 and IL-15, perhaps because of their ability
to maintain Bcl-2. In contrast to previous models in the literature,
which suggest that Bim levels are increased in response to cytokine withdrawal (36), we find just the opposite: that Bim levels are
positively correlated with cytokine availability. In the cytokine
withdrawal models, the forkhead box subclass O transcription
factor 3a (Foxo3a) has been shown to be a major transcriptional
regulator of Bim (37). The data suggest that Foxo3a is maintained
in the cytosol by cytokine signaling through Akt, and upon cytokine withdrawal, Foxo3a translocates to the nucleus and drives
expression of Bim (37, 38). However, in primary T cells, the role
for Foxo3a in controlling Bim expression is less clear. For example, one report suggested that Bim levels were not affected in
primary T cells from Foxo3a-deficient mice (39). Recently, another group reported that levels of phosphorylated (and hence
transcriptionally inactive) Foxo were actually higher in KLRG1low
CD127high CD8+ T cells relative to KLRG1highCD127low CD8+
T cells when stimulated with IL-15 (40). As KLRG1lowCD127high
CD8+ T cells have significantly higher levels of Bim compared
with KLRG1highCD127low T cells, these data suggest that Foxos
are not a significant regulator of Bim in KLRG1lowCD127high
CD8+ T cells. In contrast, another group showed that TCR and
cytokine signaling control expression of Bim in human memory
CD4+ T cell subsets through an Akt/Foxo3a-dependent mechanism (41). Although this study showed decreased Bim expression
in CD4+ TCM, our data clearly show that Bim levels in CD8+ TCM
are increased. Whether these differences are due to the differences
between humans and mice or between CD4+ and CD8+ T cells
remain to be determined. Nonetheless, more work is necessary to
further understand the regulation of Bim in effector and memory
T cell subsets.
The similarity in Bim and Bcl-2 expression between effector
and memory CD8+ T cell subsets is intriguing. At first glance,
the simplest explanation for our data would be that CD8+ TEM
cells are derived largely from KLRG1highCD127low cells, whereas
KLRG1lowCD127high cells give rise to CD8+ TCM. Indeed, the few
viral-specific CD62Lhi cells that are present shortly after the contraction of the response are located within the KLRG1lowCD127high
cell population (data not shown and Ref. 42). In addition, adoptive
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(Supplemental Fig. 1). However, as Bcl-2 can physically interact
and stabilize Bim, we decided to assess Bim levels in mice in
which T cells were deficient in Bax and Bak, thereby removing the
death signaling apparatus downstream of Bim. Interestingly, we
found that the levels of Bim were dramatically higher in both
KLRG1highCD127low and KLRG1lowCD127high effector CD8+
T cells in LckCre+Baxf/fBak2/2 mice compared with littermate
Cre2 or C57BL/6 controls (Fig. 6C). Bim mRNA levels were also
increased in effector CD8+ T cells from LckCre+ Baxf/fBak2/2
mice (Fig. 6D). Importantly, increased levels of Bim were not
accompanied by an increase in Bcl-2; in fact, Bcl-2 levels were
significantly decreased in effector CD8+ T cells from Cre+ Baxf/f
Bak2/2 mice compared with C57BL/6 mice (Fig. 6E). Moreover,
we found that the total numbers of KLRG1highCD127low and
KLRG1lowCD127high effector CD8+ T cells were significantly increased in the absence of Bax and Bak (Fig. 6F). Finally, turnover
of Bim was not significantly affected by the loss of Bax and Bak
(data not shown). Together, these data strongly suggest that
a major role for Bcl-2 is to inhibit cell death and that the level of
Bcl-2 within the cell determines the amount of Bim that T cells
can tolerate and survive.
5735
5736
Acknowledgments
We thank Drs. Edith Janssen and Claire Chougnet for critical review of the
manuscript. We also thank Dr. Aaron Johnson for assistance in generation
of Db-gp33 monomers and Dr. Stanley Korsmeyer for the Lck-Cre-Baxfl/fl
Bax2/2 mice.
Disclosures
The authors have no financial conflicts of interest.
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transfer studies have suggested that CD8+ TCM are derived from
KLRG1lowCD127high cells (12). However, the origin of CD8+ TEM
is a little less clear. The ability to track and monitor effector CD8+
T cells based on their level of Bim and/or Bcl-2 we think would
be helpful in determining the origins of memory T cell subsets.
Unfortunately, as Bim and Bcl-2 are intracellular molecules, their
levels cannot be detected while maintaining cell viability without
reporter mice.
Our results also suggest that the regulation of effector CD8+
T cell responses is dictated by a dynamic interplay carefully
controlled by the levels of pro- and antiapoptotic Bcl-2 family
members. Why would this balance be so critical? There are few
circumstances in nature whereby a quiescent cell is stimulated to
undergo 15–20 rounds of vigorous division and then return to
quiescence. During an immune response, expanded naive T cells
bear many hallmarks of neoplasia: rapid proliferation, generation
of self-renewing cells, and massive epigenetic changes. This balance of a tumor suppressor (Bim) with a proto-oncogene (Bcl-2)
is likely part of a highly conserved mechanism to prevent effector T cells from turning into T cell lymphomas. As such, we
have found that a highly effective anticancer therapeutic can
dramatically curtail effector and memory T cell responses in vivo.
Further, we found that the effects of ABT-737 on effector T cell
responses are long-lived, impacting the memory compartment
long after cessation of the drug. Thus, we envision one potential
application of Bcl-2 antagonists could be to target and eliminate
autoreactive T cells in autoimmune disease or eliminate alloreactive T cells during organ transplant. In fact, during the course
of our studies, it was shown that ABT-737 can protect mice from
allograft rejection and prevent diabetes in otherwise susceptible
mice (43). Therefore, Bcl-2 family member antagonists offer
substantial promise as a way to delete populations of unwanted,
disease-causing T cells.
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